Conventional PCR is unequivocally sufficient for the detection of "single-copy" sequences of nucleic acid, such as those represented in the genomic or chromosomal DNA of human cells, since the source material is usually derived from hundreds to thousands of cells. Unfortunately, when conventional PCR is employed in the detection of rare target sequences such as from viruses and other low-level infectious agents, or a few cancer cells among a large population of normal cells, the target sequences oftentimes remain undetectable or indeterminate (negative or borderline amplification signal). As such, nested PCR procedures have been developed to increase the amplifying power of conventional PCR manyfold, thereby greatly enhancing the sensitivity of detection of rare target nucleic acid sequences such as from the HIV provirus, cancer cells, or the like.
As is well known in the art, conventional nested PCR procedures utilize two sequential amplification processes. Specifically, the two sequential amplification processes include a first amplification process comprising at least one amplification step for amplifying an extended target sequence, and a second, subsequent amplification process comprising at least one amplification step for amplifying an internal sequence from the product of the first amplification process, wherein the internal sequence may or may not overlap one of the ends of the extended sequence. Each amplification step of the first amplification process employs an outer primer set typically comprising a pair of outer primers. Similarly, each amplification step of the second amplification process employs an inner primer set typically comprising a pair of inner primers. The above-described techniques of conventional nested PCR have been well known for almost a decade as evidenced by the Mullis et al. article "Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction", in METHODS IN ENZYMOLOGY, Academic Press, Vol. 155, 1987, pp. 335-350, incorporated herein by reference. The two sequential amplification processes of the nested PCR procedure detailed above are utilized in the present invention.
The enhanced sensitivity of nested PCR is achieved by carefully controlling the reaction conditions for the first and second amplification processes to favor the generation of the desired product. In addition to the usual considerations, most nested PCR procedures require a severalfold excess of inner over outer primers in the second amplification process for satisfactory results. Conventional nested PCR procedures generally accomplish this by amplifying only a small aliquot of the completed first amplification mixture after transfer to a new reaction tube for the second amplification process. Unfortunately, the greatly enhanced sensitivity provided by conventional nested PCR procedures is bought at the price of potential false positives, because the reaction tubes containing high concentrations of the first amplification product must be opened and manipulated to set up the second amplification, thereby increasing the probability of contamination.